Neutrophil Depletion Changes the N-Glycosylation Pattern of IgG in Experimental Murine Sepsis

Sepsis is a life-threatening condition with a rising disease burden worldwide. It is a multifactorial disease and is defined as a dysregulated host response to infection. Neutrophils have been shown to be involved in the pathogenesis of sepsis by exacerbating inflammation. However, the exact effector mechanism of action still remains a mystery. Changes in the glycosylation pattern of the immunoglobulin G (IgG) Fc region are described for several diseases including meningococcal sepsis. In this study, we investigated the possible contribution of neutrophils and neutrophil implication, potentially related to degranulation or neutrophil extracellular trap (NET) formation in changing the IgG Fc N-glycosylation pattern in a murine sepsis model. We have measured the serum level of cytokines/chemokines and immunoglobulins, the serum activity of neutrophil elastase (NE), and analyzed the IgG Fc glycosylation pattern by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) and Lectin enzyme-linked immunosorbent assay (ELISA). We observed an increased activity of NE- and neutrophil-associated cytokines such as keratinocyte chemoattractant (KC) with the development of sepsis. Regarding the IgG Fc N-glycosylation, we observed an increase in fucosylation and α1,3-galactosylation and a decrease for sialyation. Interestingly, these changes were not uniform for all IgG subclasses. After depletion of neutrophils, we saw a change in the exposure of fucose and α2,6-linked sialic acid during the time course of our experimental sepsis model. In conclusion, neutrophils can influence changes in the IgG glycosylation pattern in experimental sepsis.
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A biocompatible NIR squaraine dye and dye-antibody conjugates for versatile long-term in vivo fluorescence bioimaging

The demand for dependable near-infrared (NIR) probes, capable of sustained fluorescence within living systems and facile conjugation with biomolecules like antibodies and proteins, has been significantly on the rise, attributed to the substantial rise in the use of NIR imaging techniques and devices, with extensive integration into clinical diagnostics. Antibody conjugates are vital for targeted and selective bioimaging, enabling precise visualization of specific biomolecules within complex biological systems. Their multiplexing capability allows simultaneous detection of multiple targets, while their dynamic imaging capability enables real-time monitoring of cellular processes. Clinically, antibody conjugates have significant applications in disease prognosis, diagnosis, and monitoring. In this work, we report the synthesis of a new symmetrical NIR squaraine dye (SQ-58) with multiple carboxy anchoring groups for ease of coupling with antibodies. The dye showed decreased absorption and fluorescence intensity in phosphate buffer (PB) due to enhanced dye-aggregate formation. However, in the presence of bovine serum albumin (BSA) in PB, SQ-58 showed an enhanced fluorescence signal along concentrations of BSA. SQ-58 showed no cytotoxicity when tested in white laboratory mice while providing strong fluorescence when injected in vivo. Conjugation of SQ-58 through the carboxylic groups to the isotypic mouse IgG antibodies (IgG-SQ-58) resulted in uniform distribution of the targeted molecule in the whole cardiovascular system. The NIR signal of IgG-SQ-58 was stable for at least 7 days allowing the possibility of long-term imaging. Conjugation of SQ-58 to antibodies raised against NK-Ly lymphoma tumor cells allowed efficient discrimination of tumor cells grown in the abdomen of laboratory mice. Thus, to the best of our knowledge, we report for the first time a biocompatible NIR dye, SQ-58, that can be easily conjugatable to biomolecules, and its antibody conjugates for a wide range of bioimaging applications.

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